One aspect of the current invention involves nucleic acid ligands that inhibit an activity of lactamase enzymes, wherein the lactamase is a bacterial Class B, metallo-β-lactamase. In a preferred embodiment, a specific 30 mer nucleic acid ligand is used to inhibit a B. cereus 5/B/6 metallo-β-lactamase. Another preferred embodiment includes a specific 11 mer nucleic acid ligand is used to inhibit a B. cereus 5/B/6 metallo-β-lactamase.
Since the discovery of penicillin, β-lactam antibiotics are among the most prescribed antibacterial chemotherapeutic agents against the treatment for infectious diseases (Maugh, 1981). β-lactam antibiotics, which also include cephalosporins, monobactams and carbapenems, are analogs of peptidoglycans that are involved in the bacterial cell wall synthesis. β-lactam antibiotics target DD-peptidases (D-analyl-D-alanine carboxypeptidases/transpeptidases) that form the peptide cross-links of the peptidoglycan in the final stages of the bacterial cell wall synthesis; this takes place on the external surface of the cytoplasmic membrane and is easily accessible for the antibacterial agents. The β-lactam antibiotics inhibit DD-peptidases by forming a rather stable covalent acylenzyme complex with the DD-peptidases that has a much longer half-life than that formed with the peptidoglycan, thus disrupting the construction of the bacterial cell walls and leading to death of the bacteria (Kelly et al., 1988; Ghuysen, 1988). Because mammalian cells have a different membrane with no cell wall, β-lactams are highly specific for bacteria and even at high concentrations of β-lactams, mammalian cells are not affected.
β-lactam antibiotics all share the presence of the β-lactam ring, a four-membered ring in which a carbonyl and a nitrogen are joined in an amide linkage. FIG. 1 shows the general structures of two classes of β-lactam antibiotics: penicillin and cephalosporin. β-lactam and the adjacent atoms have similar spatial configuration to that of peptidoglycan compounds. The comparison of the structure of penicillins with the structure of D-alanyl-D-alanine-peptidoglycan are shown in FIG. 2. (Suskovic et al., 1991).
A major mechanism of resistance to β-lactam antibiotics is the production of β-lactamases (β-lactamhydrolyases, EC 3.5.2.6). β-lactamases are highly efficient enzymes that catalyze the hydrolysis of the β-lactam rings, thus rendering the loss of bactericidal activity of β-lactam antibiotics. The catalysis of hydrolysis for a generic β-lactam by a β-lactamase is shown in FIG. 3 (Livermore, 1991).
Genes for the production of β-lactamases are widely distributed among bacteria and an increasing number of pathogenic species are found to have developed multiple-drug resistance. Overcoming β-lactamases are of obvious clinical importance and studies of the mechanisms of β-lactamases are vital in the development in new β-lactam antibiotics and β-lactamase inhibitors. Slight alterations in the structures of existing β-lactam antibiotics have been utilized in response to the spread of bacterial drug-resistance; for example, cephalosporins have passed through four generations (Maugh, 1981; Pitout et al., 1997). The use of β-lactam/β-lactamase inhibitor combinations has also increased. There are limits on the chemical manipulation of the existing groups of antibiotics and it is increasingly important to design new types of antibiotics and mechanism-based β-lactamase inhibitors. Because the rational design of a β-lactam antibiotic or β-lactamase inhibitor requires a detailed understanding of the function of β-lactamases, there is great interest in the study of the mechanisms of β-lactamases.
Classification of β-lactamases. A wide range of bacteria produces β-lactamases; an incomplete list of bacteria that produce β-lactamases include Bacillus cereus, Bacillus fragilis, Escherichia coli, Aeromonous hydrophilia, Bacteroides, Staphylococcus epidermidis, Streptococcus, Pseudomonas aeruginsa, Providencia, Haemophilus, Xanthomonas maltophilia, Acinetobacter, Citrobacter, Enterobacter and Branhamella (Danziger and Pendland, 1997). There are four classes of β-lactamases: class A, B, C and D (Ambler, 1980; Ambler et al., 1991; Joris et al., 1991; Frere, 1995). Class A, C and D β-lactamases are active-site serine enzymes that resemble serine proteases and form an acyl-enzyme intermediate with an active-site serine during the catalysis of β-lactam antibiotics (Rahil and Pratte, 1991). Class A β-lactamases are soluble enzymes as are class D; however, class C β-lactamases are membrane-bound (Hussain, Pastor and Lampen, 1987). Class D β-lactamases do not exhibit primary sequence similarities to class A and C enzymes (Joris et al., 1991; Ledent et al., 1993). Class B β-lactamases are quite different from the other classes; these are metallo-β-lactamases which require divalent metal ion for enzymatic activity (Ambler, 1980; Abraham and Waley, 1979). Native class B enzymes have been isolated with one or two zinc ion(s) bound to their active sites (Carfi et al., 1995; Concha et al., 1996).
The characteristic feature of the substrate profile of class B β-lactamases is that a wide variety of β-lactam antibiotics are hydrolyzed at comparable rates while the other classes of β-lactamases have narrower substrate spectra (Abraham and Waley, 1979). β-lactam antibiotics that are substrates of class B β-lactamases include penicillin derivatives and cephalosporin derivatives (Felici et al., 1993). β-lactam antibiotics, such as carbapenems, cephamycins and imipenems, which are resistant to the serine β-lactamases are hydrolyzed by the class B β-lactamases (Felici et al., 1993; Rasmussen et al., 1994). The inhibitors for other classes of β-lactamases such as penem, 6-β-iodopenicillanic acid and penicillinic acid sulfone, do not inhibit class B β-lactamases (Felici and Amicosante, 1993). A series of mercaptoacetic acid thiol esters (Payne et al., 1997; Yang and Crowder, 1999) and thiomandelic acid (Mollard et al., 2001) have been identified as metallo-β-lactamase inhibitors and understanding the structure and dynamics of metallo-β-lactamases has been studied (Carfi et al., 1995; Concha et al., 1996; Scrofani et al., 1999). However, there is still a need to develop more effective inhibitors of metallo-β-lactamases as these enzymes have been detected in an increasing number of pathogenic bacteria (Neu, H., 1992; Payne et al., 1997).
Metallo-β-lactamase from Bacillus cereus 5/B/6. The metallo-β-lactamase was first identified in B. cereus 569. It was shown that a part of the cephalosporinase activity in the crude penicillinase preparation from B. cereus 569 required Zn2+ for maximum activity. The enzyme has unique thermal stability; heating at 60° C. for 30 min. does not abolish the catalytic activity (Crompton et al., 1962; Davies et al., 1974). The first purified metallo-β-lactamase was obtained in a protein-carbohydrate complex (Kuwabara, Adams and Abraham, 1970) from B. cereus 569/H, a spontaneous mutant of strain 569 that produces class B β-lactamase constitutively (Kogut et al., 1956). The protein purification was later modified to separate carbohydrate from the protein by gel filtration chromatography (Kuwabara and Lloyd, 1971).
Another B. cereus strain 5/B was found to produce one class A β-lactamase and one metallo-β-lactamase; this metallo-β-lactamase is very similar to the metallo-β-lactamase produced by B. cereus 569 and 569/H but with slightly different substrate specificity (Crompton et al., 1962). B. cereus 5/B/6, a mutant form of B. cereus 5/B, only produces the metallo-β-lactamase due to a mutation in the structural gene required for the synthesis of the class A β-lactamase (Davies et al., 1975; Abraham and Waley, 1979). The metallo-β-lactamase from B. cereus 5/B/6 was later purified in a similar manner from B. cereus 569/H/9 (Thatcher, 1975).
B. cereus 569/H/9 and 5/B/6 constitutively produce and secrete large amounts of metallo-β-lactamases and these enzymes, which are isolated with Zn2+ at the active site, are among the best-studied class B enzymes (Ambler, 1986; Bicknell et al., 1986; Sutton et al., 1987; Meyers and Shaw, 1989). The metallo-β-lactamases from these two strains are very similar; they both consist of 227 amino acid residues, among which 209 residues are identical (Lim, Pene and Shaw, 1988). Although these β-lactamases are isolated with Zn2+ bound at the active site, some other metal ions including Co2+, Cd2+, Mn2+, Hg2+ and Cu2+ support some catalytic activity of the enzyme (Davies and Abraham, 1974; Hilliard and Shaw, 1992; Hilliard, 1995).
The metallo-β-lactamase from B. cereus 5/B/6 has a 29 amino acid leader sequence before it is secreted from the cell. The gene for this enzyme has been cloned, sequenced, and characterized in great detail in E. coli. It has also been expressed as an intracellular enzyme with the signal sequence at relatively low levels in E. coli; it was also revealed that the metallo-β-lactamases from B. cereus strains 5/B/6 and 569/H/9 differ by 18 amino acid residues (Lim, Pene and Shaw, 1988). Even though the procedure for production and purification of metallo-β-lactamase from B. cereus 5/B/6 was greatly improved (Meyers and Shaw, 1989), hyperexpression in E. coli was still desirable. The cause of the low levels of expression was postulated to be the presence of the 29 amino acid leader peptide at the 5′-end which signals the secretion of the enzyme from B. cereus cell (Shaw et al. 1991).
Site-directed mutagenesis was performed to remove the leader sequence and to introduce a NdeI restriction endonuclease site at the initiator codon of the B. cereus 5/B/6 β-lactamase structural gene (Shaw et al., 1991); this resulted in the B. cereus 5/B/6 β-lactamase structural gene to be in a fragment between a NdeI and a SacI site. This construct allowed the cloning of the B. cereus 5/B/6 β-lactamase structural gene into the E. coli expression vector pRE2 (Reddy, Peterkofsky and McKenney 1989); this plasmid is denoted at pRE2/b1a. The recombinant plasmid pRE2 was chosen because a gene cloned into its unique NdeI and SacI restriction endonuclease sites within its polylinker region is under the control of its strong λ PL promoter. In the E. coli MZ-1, the temperature sensitive cI repressor binds to the PL promoter and prevents the expression of the B. cereus 5/B/6 β-lactamase gene on plasmid pRE2/bla at low temperatures. At higher temperatures, the cI protein is denatured, thus, allowing the expression of B. cereus 5/B/6 β-lactamase at high levels. Subsequent purifications of wild type and mutant B. cereus 5/B/6 β-lactamases resulted in a high yield of the metallo-β-lactamases which were identical (Meyers and Shaw 1989; Shaw et al., 1991).
SELEX and enzyme inhibition. In vitro selection, in vitro evolution, and Systematic Evolution of Ligands by Exponential Enrichment (“SELEX”) are common names for a technique which allows the simultaneous screening of a large number of nucleic acid molecules for different functionalities. In SELEX, large random pools of nucleic acids can be screened for a particular functionality, such as the binding to small organic molecules (Klug and Famulok, 1994), large proteins (Tuerk and Gold, 1990) or the alteration or de novo generation of ribozyme catalysis (Robertson and Joyce, 1990; Bartel and Szonstale, 1993). Functional molecules are selected from a mainly non-functional pool of RNA or DNA by column chromatography or other selection techniques that are suitable for the enrichment of any desired property.
U.S. Pat. No. 5,637,459 entitled “Systematic Evolution of Ligands by Exponential Enrichment: Chimeric Selex” issued on Jun. 30, 1998 with Burke et al., listed as inventors, and U.S. Pat. No. 5,773,598 entitled “Systematic Evolution of Ligands by Exponential Enrichment: Chimeric Selex” issued on Jun. 30, 1998 with Burke et al., listed as inventors, both of these patents describe and elaborate on the SELEX process in great detail. Both cited patents are herein incorporated by reference. Included are targets that can be used in the process; methods for partitioning nucleic acids within a candidate mixture; and methods for amplifying partitioned nucleic acids to generate enriched candidate mixture. The SELEX Patents also describe ligands obtained to a number of target species, including both protein targets where the protein is and is not a nucleic acid binding protein.
SELEX method is conceptually straightforward. A starting, degenerate oligonucleotide pool is generated using a standard DNA-oligonucleotide synthesizer; the instrument synthesizes an oligonucleotide with a completely random base-sequence, which is flanked by defined primer binding sites. The immense complexity of the generated pool justifies the assumption that it may contain a few molecules with the correct secondary and/or tertiary structures that bind tightly and specifically to a target enzyme and inhibit the enzymatic activity. These molecules are selected, for example, by affinity chromatography or filter binding. Because a large, random pool can be expected to contain only a very small number of functional molecules, several purification steps are required. Only “active” molecules are amplified by polymerase chain reaction (PCR). Iterative cycles of selection are carried out for successive selection and amplification cycles in the exponential increase in the abundance of functional sequences, until they dominate the population.
A pool of 88-mer oligonucleotides containing an internal 30-nucleotide random sequence were synthesized; this would give a possibility of 430 (1.2×1018) different sequences projected from the possibility of any four nucleotides in 30 positions. The internal 30-nucleotide region is flanked by defined primer sites which are 43 and 15 nucleotides at their 5′ and 3′ termini, respectively. These were later transcribed and the single-stranded RNA has been selected that not only binds tightly and specifically to the B. cereus 5/B/6 metallo-β-lactamase but also inhibits this enzyme.
Prediction of secondary structure of aptamers. MFold program is an adaptation of the MFold package (version 2.3) by Zuker (1989) and Jaeger et al. (1989, 1990) that has been modified to work with the Wisconsin Package™. Their method uses the energy rules developed by Freier et al. (1986) to predict optimal secondary structures for an RNA molecule and the energy rules complied and developed by Turner et al. (1988) to predict optimal and sub-optimal secondary structures for a single-stranded RNA molecule. This approach can provide a first-approximation prediction of a nucleic acid secondary structure from a nucleic acid sequence.
The invention described herein has utilized the method of SELEX to develop nucleic acid ligands for a lactamase enzyme. The nucleic acid ligands are utilized as metallo-β-lactamase inhibitors.